Visualizing Presynaptic Calcium Dynamics and Vesicle Fusion with a Single Genetically Encoded Reporter at Individual Synapses.

Rachel E Jackson,Juan Burrone

doi:10.3389/fnsyn.2016.00021

Abstract

Synaptic transmission depends on the influx of calcium into the presynaptic compartment, which drives neurotransmitter release. Genetically encoded reporters are widely used tools to understand these processes, particularly pHluorin-based reporters that report vesicle exocytosis and endocytosis through pH dependent changes in fluorescence, and genetically encoded calcium indicators (GECIs) that exhibit changes in fluorescence upon binding to calcium. The recent expansion of the color palette of available indicators has made it possible to image multiple probes simultaneously within a cell. We have constructed a single molecule reporter capable of concurrent imaging of both presynaptic calcium influx and exocytosis, by fusion of sypHy, the vesicle associated protein synaptophysin containing a GFP-based pHluorin sensor, with the red-shifted GECI R-GECO1. Due to the fixed stoichiometry of the two probes, the ratio of the two responses can also be measured, providing an all optical correlate of the calcium dependence of release. Here, we have characterized stimulus-evoked sypHy-RGECO responses of hippocampal synapses in vitro, exploring the effects of different stimulus strengths and frequencies as well as variations in external calcium concentrations. By combining live sypHy-RGECO imaging with post hoc fixation and immunofluorescence, we have also investigated correlations between structural and functional properties of synapses.

Highlights

Synaptic transmission depends on the influx of calcium into the presynaptic compartment, driving the release of vesicles containing neurotransmitter, which will bind to receptors on the postsynaptic cell
To generate a single molecule reporter of both presynaptic calcium influx and synaptic vesicle exocytosis, the R-GECO1 fragment was amplified from SyRGECO (Walker et al, 2013) and subcloned downstream of sypHy (Granseth et al, 2006) with a short linker sequence
Presynaptic calcium influx is reported by an increase in red fluorescence and exocytosis of synaptic vesicles by an increase in green fluorescence (Figure 1A)

Summary

Introduction

Synaptic transmission depends on the influx of calcium into the presynaptic compartment, driving the release of vesicles containing neurotransmitter, which will bind to receptors on the postsynaptic cell. Optical strategies using either chemical indicators or genetically encoded reporters have become the method of choice. The largest family of single color genetically encoded calcium indicators (GECIs) is the GCaMP family, based on a circularly permuted GFP linked to calmodulin (CaM) and it’s binding peptide M13 (Nakai et al, 2001). To enable multicolor imaging in single cells, red-shifted GECIs have been developed, including RCaMP (Akerboom et al, 2013) and R-GECO1 (Zhao Y. et al, 2011), for which a presynaptically targeted version (SyRGECO) has been characterized (Walker et al, 2013). Much like the GCaMPs, red GECIs are constantly evolving, with new variants recently produced in both RCaMP and R-GECO families (Inoue et al, 2015; Dana et al, 2016)

Methods

Results

Conclusion